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Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells
Accepted Manuscript Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells
Jun Ying, Pinger Wang, Shanxing Zhang, Taotao Xu, Lei Zhang, Rui Dong, Shibing Xu, Peijian Tong, Chengliang Wu, Hongting Jin PII: DOI: Reference:
S0024-3205(17)30605-7 doi:10.1016/j.lfs.2017.11.028 LFS 15440
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Life Sciences
Received date: Revised date: Accepted date:
25 September 2017 12 November 2017 16 November 2017
Please cite this article as: Jun Ying, Pinger Wang, Shanxing Zhang, Taotao Xu, Lei Zhang, Rui Dong, Shibing Xu, Peijian Tong, Chengliang Wu, Hongting Jin , Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/ j.lfs.2017.11.028
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ACCEPTED MANUSCRIPT Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells Jun Ying1,2¶, Pinger Wang2¶, Shanxing Zhang3, Taotao Xu1,2, Lei Zhang1,2, Rui Dong1,2, Shibing Xu1,2, Peijian Tong3, Chengliang Wu1,2*, Hongting Jin1,2,3* 1
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¶These authors contributed equally to this work.
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First Clinical College of Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China 2 Institute of Orthopaedics and Traumatology, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China 3 Department of Orthopaedic Surgery, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, Zhejiang Province, China
*Corresponding author
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Hongting Jin, M.D. Ph.D Institute of Orthopaedics and Traumatology The First Affiliated Hospital of Zhejiang Chinese Medical University No.548, Binwen Road, Hangzhou, 310053 Zhejiang, China Tel: 011-86 571-86633057; Fax: 011-86 571-86613684 Email: [[email protected]] Or Chengliang Wu, M.D. Ph.D Institute of Orthopaedics and Traumatology The First Affiliated Hospital of Zhejiang Chinese Medical University No.548, Binwen Road, Hangzhou, 310053 Zhejiang, China Tel: 011-86 571-86633057; Fax: 011-86 571-86613684 Email: [[email protected]]
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Abstract Aims: Transforming growth factor-β1 (TGF-β1) is a chondrogenic factor and has been reported to be able to enhance chondrocyte differentiation from bone marrow mesenchymal stem cells (BMSCs). Here we investigate the molecular mechanism through which TGF-β1 chronically promotes the repair of cartilage defect and inhibit chondrocyte hypertrophy. Main methods: Animal models of full thickness cartilage defects were divided into three groups: model group, BMSCs group (treated with BMSCs/calcium alginate gel) and BMSCs + TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transduced BMSCs/calcium alginate gel). 4 and 8 weeks after treatment, macroscopic observation, histopathological study and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were done to analyze phenotypes of the animals. BMSCs were transduced with Lentivirus-TGF-β1-EGFP in vitro and Western blot analysis was performed. Key findings: We found that TGF-β1-expressiing BMSCs improved the repair of the cartilage defect. The impaired cartilage contained higher amount of GAG and type II collagen and was integrated to the surrounding normal cartilage and higher content of GAG and type II collagen. The major events include increased expression of type II collagen following Smad2/3 phosphorylation, and inhibition of cartilage hypertrophy by increasing Yes-associated protein-1 (YAP-1) and inhibiting Runx2 and Col10 after the completion of chondrogenic differentiation. Significance: We conclude that TGF-β1 is beneficial to chondrogenic differentiation of BMSCs via canonical Smad pathway to promote early-repairing of cartilage defect. Furthermore, TGF-β1 inhibits chondrocyte hypertrophy by decreasing hypertrophy marker gene expression via Hippo signaling. Long-term rational use of TGF-β1 may be an alternative approach in clinic for cartilage repair and regeneration.
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Keywords Transforming growth factor-β1; Bone marrow mesenchymal stem cells; Smad signaling; Hippo signaling; Cartilage defect
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Introduction Cartilage injury and degeneration is common with the development of aging and trauma (Madry et al. , 2016). Unfortunately, due to lack of vascularity and the repair cells, poor capacity for self-repair of cartilage is hard to change without external treatments after injury (Park et al. , 2017, van den Borne et al. , 2007). Osteoarthritis (OA), a common degenerative joint disease, is often associated with cartilage defect (Chen et al., 2016). Currently, drug-related treatments are confined to the improvement of OA symptoms such as pain and inflammation (Santaguida et al. , 2008). Microfracture, autologous chondrocyte implantation (ACI) and osteochondral autograft have also been tried to treat OA, with unsatisfactory effects (Park et al., 2017). MSCs-based therapies have a great promise to regenerate cartilage defects (Richardson et al. , 2010). Because of the extraordinary potential for proliferation and multipotential differentiation including chondrogenesis (Jin et al. , 2016), BMSCs have been widely studied in the mechanisms of articular cartilage repaired. However, according to the previous studies, direct application of MSCs is not efficient for their propensity to lose chondrogenic phenotype. In order to avoid inefficiency and premature degeneration, chondrogenic-related growth factors are crucial for directing and maintaining chondrogenesis in a microenvironment (Choi et al. , 2015). TGF-β1, a chondrogenesis-inducing factor, is crucial for chondrogenic differentiation of BMSCs (Shen et al., 2013; 2014). By using TGF-β1-supplemented medium, Johnstone et al. had firstly induced differentiation of human BMSCs into chondrogenic lineages (Johnstone et al. , 1998). So many scholars has been attracted to investigated the mechanism between TGF-β1 and chondrogenesis of MSCs. TGF-β1 can facilitate proliferation and maturation of chondrocytes, and improve aggregation of type II collagen and proteoglycan(Liu et al. , 2015). Tissue engineering approach, which contains isolated seed cells, degradable scaffolds and certain growth factors, seems to provide an optional solution to restore cartilage. Alginate [(C6H8O6)n], a natural polysaccharide extracted from brown algae, composed of β-D-mannuronate (M) and α-L-guluronate (G) (Growney Kalaf et al. , 2016, Lee and Mooney, 2012). It has been widely used to provide structures for transporting cells and bioactive molecules as a harmless biomaterial with lack of toxicity or immunogenicity. The Hippo pathway plays a key role in tissue regeneration(Hong et al. , 2016).And YAP, a key transcriptional co-activator, has been implicated recently in regulation of chondrogenesis, which provides new enlightenment for cartilage-repairing (Zhong et al. , 2013). Deng et al. (Deng et al. , 2016) found that Yap1 can inhibit chondrocyte hypertrophy by inhibiting Col10al expression through interaction with Runx2, a crucial regulator of chondrocyte hypertrophy. TGF-β1 can also inhibit the growth of hepatocellular carcinoma (HCC) cells by regulating the Hippo signaling pathway (Zhang et al. , 2017). And TGF-β1 in Human Embryonic Stem Cells can enhance the interactions of YAP1 and Smad to regulate cellular responses(Beyer et al. , 2013). However, it is still not clear the interactions and
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functions of the TGF-β1 and Hippo signaling pathway in the chondrogenesis of BMSCs and full-thickness articular cartilage repair. Besides, lentiviral is an optional transgenic vector due to the high infection efficiency and stable expression of target-genes (Naldini et al. , 1996). In the present study, Lentivirus-TGF-β1-EGFP was transfected in BMSCs, which were uniformly encapsulated in calcium alginate. Then BMSCs/calcium alginate constructs were implanted to cartilage defect sites in rat models. It was hypothesized that the Lentivirus-TGF-β1-EGFP transfection can enhance chondrogenesis of BMSCs to promote early-repairing of cartilage defect by TGF-β1-dependent Smad2/3 signaling and then regulate Hippo signaling to inhibit chondrocyte hypertrophy in surgically-induced full-thickness articular cartilage.
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Materials and Methods 1. Isolation and culture of BMSCs This study was approved by the Animal Care and Use Committee in Zhejiang Chinese Medical University. BMSCs were isolated from the Sprague-Dawley rats(male, 4-week-old) in a sterile environment. The bilateral femoral shafts were extracted and washed with sterile Phosphate-Buffered Saline (PBS). After removing the metaphysis to expose the bone marrow cavity, they were flushed with 10 mL serum free Dulbecco's modified eagle medium (DMEM) by 5ml syringe. Cell suspension was filtered through a 200-mesh cell sieve and centrifuged for 5min at 1000rpm. Then the cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and incubated at 37 °C and 5% CO2. By the differential time adherence method, BMSCs were separated from cells mixture. Then cells were digested using EDTA-Trypsin until reaching about 90% confluence. BMSCs were cultured and purified for three generations.
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2. Lentivirus transfection of BMSCs With the multiplicity of infection (MOI) of 100, Lentivirus-EGFP and Lentivirus-TGF-β1-EGFP (Shanghai Genechem Co., Ltd., Shanghai, China) were respectively transfected in third passage BMSCs, added with 5μl/ml polybrene. Green fluorescence was observed after 72h by using a fluorescence microscope (Olympus IX2-ILL100; Olympus Corp., Tokyo, Japan). With the cultivation in the medium above, BMSCs were divided into three groups in vitro experiment : control group(no transfection); vector group(with Lentivirus-EGFP transfection) and TGFβ1 group(with Lentivirus-TGF-β1-EGFP transfection). 3. Preparation of BMSCs/calcium alginate gel Aqueous solution of sodium alginate (2%) and calcium chloride (1%) were prepared firstly. They were sterilized by using 0.22-μm membrane filters. BMSCs, including non-transfection and Lentivirus-TGF-β1-EGFP transfection were suspended in 5ml of 2% sterile sodium alginate solution respectively at the final concentration of 1×107/ml. Then, BMSCs/sodium alginate hydrogel was dropped into calcium chloride solution to form the BMSCs/calcium alginate gel.
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4. Rat model of full thickness cartilage defect In the present study, 36 Sprague-Dawley rats (male, 12-week-old) were used. Animals were anesthetized using ketamine (80mg/kg) administered by intraperitoneal injection. With a medial parapatellar incision, the knee joints were opened to exposed the trochlear surface directly. Full thickness cartilage defects (2mm diameter 2mm deep) were drilled on the surface of trochlear groove by using an electrical drilling machine. All rats were randomly divided into three groups (n=12/group at weeks 4 and 8): model group (treated with nothing as controls), BMSCs group (treated with BMSCs/calcium alginate gel), BMSCs+TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel). After surgery, all rats were treated with postoperative antibiotic (penicillin) intramuscularly at 8U/rat for 3 days. After 4 and 8 weeks, all animals were euthanized in batches.
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5. Gross view, histology, immunohistochemistry (IHC) and histological evaluation Samples were harvested (n=6 at weeks 4 and 8) and separated freely from soft tissue and photographed for gross evaluation. International Cartilage Repair Society (ICRS) cartilage repair scores were evaluated referred to Table1 (Lee et al. , 2013).
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Table 1. ICRS cartilage repair scores Points In level with surrounding cartilage 75% repair of defect depth Degree of defect 50% repair of defect depth repair 25% repair of defect depth no repair of defect depth Complete integration with surrounding cartilage Demarcating border<1mm
Macroscopic appearance
75% repair tissue integrated, 25% with an evident border >1mm 50% repair tissue integrated, 25% with an evident border >1mm From no contact to 25% repair tissue integrated with surrounding cartilage Intact smooth surface Fibrillated surface Small, scattered fissures or cracks Several, small or few but large fissures Total degeneration of defect area
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Integration to border zone
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Standards
Scores 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0
Then they were fixed in 10% normal buffered formalin for 72h, decalcified with 14% EDTA (PH=7.4) for 30 days, dehydrated and embedded in paraffin, and cut into 3μm sections, which were stained with Alcian Blue Hematoxylin/Orange G (ABH). In order to evaluate degree of defect repair, the Mankin score system [20,21] was used.
ACCEPTED MANUSCRIPT Total 13 score was combined with structure (0-6 points), matrix (0-4 points) and cellular abnormalities (0-3 points). In the present study, lower score indicates better cartilage repair. Three independent observers performed this histopathological evaluation. IHC was performed using anti-type II collagen (Millipore, mab1330, USA), anti-pSmad2 (Abcam, ab188334, UK), anti-Runx2 (Medical & Biological Laboratories, D130-3, Japan), anti-Col10 (Abcam, ab58632, UK) and anti-YAP-1(Abcam, ab39361, UK) antibodies on the 3-μm thick tissue sections.
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6. Quantitative Gene Expression Analysis Total RNA was extracted from repaired cartilage and cells by using the RNeasy kit (Qiagen, Germany) according to the manufacturer’s instructions and measured concentration and quality by the Nanodrop 2000 (Thermo, USA). Isolated RNA was reverse transcribed to complementary DNA (cDNA) using a cDNA reverse transcription kit (Takara, Japan). QRT-PCR was performed by using an SYBR Premix EX TaqTM Ⅱ kit (Takara, Otsu, Japan). Primer sequences for GAPDH and Col2a1 are shown in Table 2.The expression level of cartilage-related genes (Col2a1) was analyzed. The ΔΔCt method was used to calculate the expression of mRNAs relative to GAPDH. All procedures were processed under RNase-free condition. Table2. Primer name and sequences for PCR analysis Primer Name Sequences GAPDH forward 5’-GAACATCATCCCTGCATCCA-3’ GAPDH reverse 5’- CCAGTGAGCTTCCCGTTCA-3’ Col2a1 forward 5’-TCCTAAGGGTGCCAATGGTGA-3’ Col2a1 reverse 5’-AGGA CCAACTTTGCCTTGAGGAC-3’
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7. Western blot Analysis Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mM phenylmethyl sulfonyl fluoride (PMSF) and protease inhibitor cocktail (Cell Signaling Technology, USA). The supernatant was collected after centrifugation at 12000 g for 30min at 4℃. Protein concentrations were detected by using a BCA Protein Assay kit (Thermo Scientific). Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, USA) incubated with different primary antibodies against β-actin (Sigma, A1978, USA), phosphorylated-Smad2/3(Cell signaling technology, #8828, USA), Smad2/3 (Cell signaling technology, #8685, USA), TGFβ1(Abcam, ab92486, UK) and YAP-1 (Abcam, ab39361, UK) overnight at 4℃. Fluorescent secondary antibodys (Licor, 926-32212 USA) were incubated with membrane in a dark room for 1h on the next day. The chemiluminescence on the membrane was detected using the he LI-COR Odyssey® scanner and software (LI-COR Biosciences, USA). 8. Statistical Analysis All data were presented as mean ± standard deviation. Statistical analyses included unpaired Student’s t-tests and one-way ANOVA test followed by the
ACCEPTED MANUSCRIPT Tukey-Kramer test. The statistical tests were performed using the software SPSS 17.0. Statistical significance was set at a 𝑃 value of <0.05.
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Results TGFβ1 is beneficial to repair cartilage defect by enhancing chondrogenesis of BMSCs. Full-thickness articular cartilage defects were made in the trochlear groove of the rat distal femur, and BMSCs/calcium alginate gel alone or transfected with TGF-β1 were implanted in the defects (Figure 1).
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Figure 1. (A) Gross views of formed BMSCs/calcium alginate gels. (B) Microscopic observation of one formed BMSCs/calcium alginate gel. Bar=2mm;.
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Macroscopically, at 4 and 8 weeks after surgery, the defect was not repaired at all in model group. Especially, friable tissue grew on the surface at weeks 8. In BMSCs group, the surface of repaired tissue was rough accompanied with a clear boundary during week 4 to 8 post-surgery. Relatively, compared to BMSCs group, the defects in BMSCs+TGFβ1 group contained more intact fillings with good integration to the surrounding normal cartilage (Figure 2A). The ICRS-cartilage repair scores in BMSCs+TGFβ1 group was significantly higher than the model group and BMSCs group (P<0.05) at 4 and 8-weeks post-op, proving better repairs than the model group and BMSCs group (Figure 2B).
ACCEPTED MANUSCRIPT Figure 2. (A)Representative gross views of repaired cartilage defects at weeks 4 and 8 in the black arrowheads. Defects were left untreated (model group) or treated with non-transfected BMSCs/calcium alginate gel (BMSCs group) and Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel (BMSCs+TGF-β1 group). (B) Evaluation of ICRS macroscopic cartilage repair scores. *P<0.05 and P<0.05 versus BMSCs+TGF-β1 group, **P<0.01 and P<0.01 versus BMSCs+TGF-β1 group.
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In order to observe the cellular structure and matrix composition between the different groups, we performed with staining of ABH and IHC for type II collagen. The histological evaluation showed parallel results. In model group, especially in 8 weeks, the defect was mainly covered with fibrous tissue. However, the defects in BMSCs+TGFβ1 group covered with more cartilage-like tissues accumulated with GAG than BMSCs group (Figure 3A). The Mankin score at weeks 4 and 8 indicated that model group were higher (8.3, 10.7) than BMSCs+TGF-β1 group (4.5, 3.5; P<0.01), and at weeks 4, BMSCs group (7.5) got higher Mankin score than BMSCs+TGF-β1 group (4.5; P<0.05). However, there is no significant difference of Mankin score between model group and BMSCs group at weeks 4(P>0.05) (Figure 3B). These data above revealed that the repaired cartilage in BMSCs+TGF-β1 group was the most similar to normal cartilage. IHC for type II collagen in each group was also performed during the weeks 4, 8 after surgery to observe chondrogenic tissue formation. The results showed that the expression of type II collagen in the repaired tissue was unobvious at both time points in model group. Repaired tissues in BMSCs group and BMSCs+TGF-β1 group stained positive for type II collagen at weeks 4 and 8 after surgery. The BMSCs+TGF-β1 group showed higher content of type II collagen than BMSCs group. By week 8, the accumulation of type II collagen in BMSCs+TGF-β1 group almost similar to the normal cartilage surrounding the defects (Figure 3C). Besides, in order to detect the cartilage-related mRNA expression of Col2a1 in the repaired tissue, quantitative real-time fluorescent PCR was used for each group. These were consistent with the histopathological results. The mRNA level of Col2a1 in BMSCs+TGF-β1 group was significantly higher than in model group and BMSCs group at the same time point (P<0.01, Figure 3D). Interestingly, Col2a1 mRNA expression in BMSCs group was higher than that in model group (P<0.01). These data above indicated that type II collagen and proteoglycan were effectively accumulated in cartilage defects treated with Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel.
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Figure 3. TGFβ1-transfected BMSCs effectively promote cartilage repairing. (A). Histological sections of repaired cartilage defects stained using Alcian Blue Hematoxylin/Orange G. Black arrowheads indicate repaired regions. Bar=3mm; (B) Mankin scores of histological repairing cartilages at defect sites; (C). Immunohistochemistry (brown) of collagen type II in repaired regions. Black arrows indicate repaired regions. Bar=1mm; (D). Total RNA was extracted from repaired tissues at weeks 4 and 8. In BMSCs+TGF-β1 group, expression of Col2a1 was significantly increased at 4 and 8 weeks. Data are presented as means ± SD. *P<0.05 and △P<0.05 versus BMSCs+TGF-β1 group, P<0.01 versus BMSCs group, **P<0.01 versus BMSCs+TGF-β1 group and P<0.01 versus model group.
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Lentivirus-TGF-β1-EGFP transfection increases activation of phosphorylation-Smad2/3 during chondrogenesis of BMSCs We analyzed TGF-β1-dependent Smad2/3 signaling activated by Lentivirus-TGFβ1-EGFP transfection to identify whether this treatment induce activation of chondrogenic differentiation through the intracellular signaling in BMSCs. IHC for pSmad2 was used to observe activation of Smad2/3 signaling during repaired tissue formation. As shown in Figure 4A, the positive cells of phospho-Smad2/3 were not apparent in Model group at the weeks 4. BMSCs alone weakly increased activation of phospho-Smad2/3. There is no significant difference between model group and BMSCs group (P>0.05). However, lentivirus-TGF-β1-EGFP transfected treatment strongly activated the phosphorylation of Smad2 at the weeks 4 (Figure 4B). It is worth noting that lentivirus-TGFβ1-EGFP transfection enhanced phospho-Smad2/3 activation in the early-period of repairing. To demonstrate the effect of the TGFβ1 on early-chondrogenic differentiation of BMSCs, we used BMSCs with non-transfected, transfected with Lentivirus-EGFP or Lentivirus-TGF-β1-EGFP and analyzed the protein expression of phospho-Smad2/3, which was specified as the TGF-β1-induced pathway to increase chondrogenic maker
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expression, such as Col2a1 (Furumatsu et al. , 2005). In vitro experiment, the Western blot results showed that the Lentivirus-TGFβ1-EGFP transfection increased phosphorylation of Smad2/3 at day 2 compared to control group and vector group (Figure 4D).
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Figure 4. Overexpression of TGF-β1 promotes the phosphorylation of Smad2/3 in BMSCs during the early repairing progress. (A). IHC of phospho-Smad2 (brown) in repaired regions. Red arrowheads: phospho-Smad2-positive cells. Bar=50μm; (B). BMSCs were observed using microscopy with normal and EGFP under the same vision at day 3 following transfection with Lentivirus-TGF-β1-EGFP. Bar=1mm; EGFP, enhanced green fluorescent protein. (C). Positive cell ratio of phospho-Smad2 was significantly increased in BMSCs+TGF-β1 group (week 4). (D). Western blot analysis demonstrated that Lentivirus-TGF-β1-EGFP was successfully transfected into BMSCs and the protein expression of pSmad2/3 was significantly increased in the BMSCs+TGF-β1 group, compared with the control and vector groups. Data are presented as means ± SD. **P<0.01 versus BMSCs+TGF-β1 group.
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TGF-β1 inhibit chondrocyte hypertrophy by regulating Hippo signaling pathway As mentioned above, TGF-β1 regulated the Hippo signaling pathway in HCC cells and YAP-1 can inhibit chondrocyte hypertrophy. In order to explore whether TGF-β1 influences the Hippo signaling pathway to inhibit hypertrophy after chondrogenic differentiation of BMSCs, we tested the expressions of YAP-1 and hypertrophic chondrocyte markers. The IHC showed that lentivirus-TGF-β1-EGFP transfected treatment obviously increased the expression of YAP-1 compared to model group and BMSCs group at the weeks 8. TGF-β1 also effectively inhibited the expressions of Runx2 and Col10 in the repaired cartilages of full-thickness articular cartilage defects rats (Figure 5A+ Figure 5B). The Western blot results in vitro also indicated that the Lentivirus-TGF-β1-EGFP transfection significantly increased YAP-1 expression at day 7 compared to control group and vector group (Figure 5C). It is proved that TGF-β1 regulated Hippo signaling pathway to inhibit chondrocyte hypertrophy by decreasing the expression of YAP-1, Runx2 and Col10.
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Figure 5. TGF-β1 decrease hypertrophic markers expression to inhibit chondrocyte hypertrophy by increasing YAP-1 expression at week8. (A). IHC of YAP-1, Runx2 and Col10 (brown) in repaired regions. (B). Positive cell ratio of YAP-1 was significantly increased in BMSCs+TGF-β1 group (week 8). (C). The protein expression of YAP-1 was significantly increased in the BMSCs+TGF-β1 group in vitro, compared with the control and vector groups; Bar=50μm; Data are presented as means ± SD. **P<0.01 versus BMSCs+TGF-β1 group.
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Discussion Clinically, cartilage damage resulted from trauma or pathologic factors is common and often reduce the patient’s quality of life (Brittberg, 2010). Most of the clinical repair methods for cartilage regeneration have favorable short-term effect, but the long-term effect is unsatisfied (Zhu et al. , 2014). Due to the poor expansion capacity of chondrocytes, autologous chondrocyte implantation is not an ideal choice for cartilage repair (Cochis et al. , 2017). In free matrix and matrix-dependent cell cultures, MSCs has shown its chondrogenic potential (Chen et al. , 2016, Johnstone et al., 1998, Noth et al. , 2002). In clinical, there are mainly two ways of using autologous MSCs to repair articular cartilage defects: intra-articular injections of MSCs (Nejadnik et al. , 2010) and surgical transplantation of MSCs-scaffold constructs (Gobbi et al. , 2014, Gobbi et al. , 2016). Tissue engineering is a promising method because it contains abundant seed cells such as MSCs which are be beneficial to cartilage repair and can avoid immunoreaction due to the source of autologous body. In this study, the BMSCs were isolated and cultured in vitro, transfected with Lentivirus-TGF-β1-EGFP and construct BMSCs/calcium alginate gel to enhance chondrogenesis of BMSCs to improve cartilage repair. We demonstrated that TGF-β1 promotes BMSCs chondrogenesis and cartilage repairing via TGF-β1-dependent Smad2/3 signaling and inhibit chondrocyte hypertrophy by regulating the Hippo signaling pathway. The results of the current study have showed the potential utility of gene-related therapy in promotion of cartilage repairing. Series of studies have proved that TGF-β1 promote MSCs to differentiate into chondrocytes, accumulate the secretion of type II collagen and proteoglycan (Liu et
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al., 2015). Our data indicate that TGF-β1 significantly enhance chondrogenesis of BMSCs in vitro and improve cartilage repairing in vivo via activation of TGF-β1-dependant Smad2/3 signaling pathway. Gross observation and histologic analysis showed that BMSCs+TGF-β1 group got the best repairing accompanied with the most smooth repairing surface, accumulation of type II collagen and proteoglycan. The results of ICRS-cartilage repair scores and Mankin score were consistent with it. The enhanced chondrogenic differentiation of BMSCs was related to increased gene expression of Col2a1. Besides, TGF-β1 can also inhibit generation of fibrous tissue in defect regions. In canonical Smad2/3 pathway, phosphorylated-Smad2 and 3 form complexes with the transcription factor Smad4. And the complexes bind specific promoters to regulate target genes (Wang et al. , 2016). Smad2/3 signaling provides equally important effects for chondrogenesis and reparative response of cartilage (Hellingman et al. , 2011). The data of Western blot and IHC proved that TGF-β1 enhances chondrogenesis of BMSCs by activating phosphorylation of Smad2/3 at the early cartilage repairing phase. Besides, Karl et al (Karl et al. , 2014) demonstrated that removed TGFβ1 from the chondrogenic medium significantly increased the hypertrophic phenotype in chondrogenic differentiating MSCs. Our data also proved that Lentivirus-TGF-β1-EGFP transfection inhibited expression of Col10a1 after chondrogenic differentiating of BMSCs during the repairing progress. Yap1, a key transcriptional co-factor in Hippo signaling pathway, promotes proliferation of early chondrocyte but interaction with Runx2, which is a crucial regulator for chondrocyte hypertrophy to suppress the expression of target genes such as Col10a1, the hypertrophy marker (Deng et al., 2016, Takeda et al. , 2001, Zheng et al. , 2003). We then analyzed whether TGF-β1 influence the expressions of hypertrophic markers of chondrogenic differentiating BMSCs via Hippo signaling pathway. We tested the expressions of YAP-1, Runx2 and Col10a1 at weeks 8 in vivo. Interestingly, the expression of YAP-1 was increased at weeks 8 when the rats were treated with lentivirus-TGF-β1-EGFP transfected BMSCs. Meanwhile, Runx2 and Col10a1 were inhibited in BMSCs+TGF-β1 group. The Western blot results in vitro also demonstrated that TGF-β1 can significantly increases YAP-1 expression after chondrogenic differentiating of BMSCs. So it means that TGF-β1 can inhibit expressions of chondrocyte hypertrophic markers via Hippo signaling. Therefore, we concluded that TGF-β1 plays two roles in cartilage repairing: 1). TGFβ1 is beneficial to chondrogenesis of BMSCs via phosphorylation of Smad2/3 to increase the expression of cartilage formation markers. 2). TGF-β1 can inhibit chondrocyte hypertrophy by decreasing hypertrophy markers expression via Hippo signaling. Conclusions We demonstrated that the implantation of Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel promoted cartilage defect repair and regeneration through chondrogenic differentiation and inhibition of hypertrophy, which are strongly associated with TGF-β1-dependent Smad2/3 signaling and Hippo signaling respectively.
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Corresponding Authors * [email protected] and [email protected] Acknowledgements This research has been partially supported by Zhejiang grants funded by Provincial Natural Science Foundation of China (Grant no. LY16H270010, LZ12H27001 and LY15H270012) Natural Science Foundation of China (Grant no.81373669, 81573994 and 81774332), the State Administration of Traditional Chinese Medicine of Zhejiang Province (Grant no. 2016ZA048, 2018ZZ011 and 2018ZA034), Major Scientific and Technological Special Project for during the Twelfth Five-year Plan Period (Grant no. 2014C13G2120082) and the Program for Zhejiang Leading Team of S&T Innovation and Key Laboratory of Zhejiang Province. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
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Jun Ying, Pinger Wang, Shanxing Zhang, Taotao Xu, Lei Zhang, Rui Dong, Shibing Xu, Peijian Tong, Chengliang Wu, Hongting Jin PII: DOI: Reference:
S0024-3205(17)30605-7 doi:10.1016/j.lfs.2017.11.028 LFS 15440
To appear in:
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Received date: Revised date: Accepted date:
25 September 2017 12 November 2017 16 November 2017
Please cite this article as: Jun Ying, Pinger Wang, Shanxing Zhang, Taotao Xu, Lei Zhang, Rui Dong, Shibing Xu, Peijian Tong, Chengliang Wu, Hongting Jin , Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/ j.lfs.2017.11.028
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ACCEPTED MANUSCRIPT Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells Jun Ying1,2¶, Pinger Wang2¶, Shanxing Zhang3, Taotao Xu1,2, Lei Zhang1,2, Rui Dong1,2, Shibing Xu1,2, Peijian Tong3, Chengliang Wu1,2*, Hongting Jin1,2,3* 1
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¶These authors contributed equally to this work.
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First Clinical College of Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China 2 Institute of Orthopaedics and Traumatology, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China 3 Department of Orthopaedic Surgery, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, Zhejiang Province, China
*Corresponding author
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Hongting Jin, M.D. Ph.D Institute of Orthopaedics and Traumatology The First Affiliated Hospital of Zhejiang Chinese Medical University No.548, Binwen Road, Hangzhou, 310053 Zhejiang, China Tel: 011-86 571-86633057; Fax: 011-86 571-86613684 Email: [[email protected]] Or Chengliang Wu, M.D. Ph.D Institute of Orthopaedics and Traumatology The First Affiliated Hospital of Zhejiang Chinese Medical University No.548, Binwen Road, Hangzhou, 310053 Zhejiang, China Tel: 011-86 571-86633057; Fax: 011-86 571-86613684 Email: [[email protected]]
Word count: 5191 Figure count: 5 Table count: 2
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Abstract Aims: Transforming growth factor-β1 (TGF-β1) is a chondrogenic factor and has been reported to be able to enhance chondrocyte differentiation from bone marrow mesenchymal stem cells (BMSCs). Here we investigate the molecular mechanism through which TGF-β1 chronically promotes the repair of cartilage defect and inhibit chondrocyte hypertrophy. Main methods: Animal models of full thickness cartilage defects were divided into three groups: model group, BMSCs group (treated with BMSCs/calcium alginate gel) and BMSCs + TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transduced BMSCs/calcium alginate gel). 4 and 8 weeks after treatment, macroscopic observation, histopathological study and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were done to analyze phenotypes of the animals. BMSCs were transduced with Lentivirus-TGF-β1-EGFP in vitro and Western blot analysis was performed. Key findings: We found that TGF-β1-expressiing BMSCs improved the repair of the cartilage defect. The impaired cartilage contained higher amount of GAG and type II collagen and was integrated to the surrounding normal cartilage and higher content of GAG and type II collagen. The major events include increased expression of type II collagen following Smad2/3 phosphorylation, and inhibition of cartilage hypertrophy by increasing Yes-associated protein-1 (YAP-1) and inhibiting Runx2 and Col10 after the completion of chondrogenic differentiation. Significance: We conclude that TGF-β1 is beneficial to chondrogenic differentiation of BMSCs via canonical Smad pathway to promote early-repairing of cartilage defect. Furthermore, TGF-β1 inhibits chondrocyte hypertrophy by decreasing hypertrophy marker gene expression via Hippo signaling. Long-term rational use of TGF-β1 may be an alternative approach in clinic for cartilage repair and regeneration.
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Keywords Transforming growth factor-β1; Bone marrow mesenchymal stem cells; Smad signaling; Hippo signaling; Cartilage defect
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Introduction Cartilage injury and degeneration is common with the development of aging and trauma (Madry et al. , 2016). Unfortunately, due to lack of vascularity and the repair cells, poor capacity for self-repair of cartilage is hard to change without external treatments after injury (Park et al. , 2017, van den Borne et al. , 2007). Osteoarthritis (OA), a common degenerative joint disease, is often associated with cartilage defect (Chen et al., 2016). Currently, drug-related treatments are confined to the improvement of OA symptoms such as pain and inflammation (Santaguida et al. , 2008). Microfracture, autologous chondrocyte implantation (ACI) and osteochondral autograft have also been tried to treat OA, with unsatisfactory effects (Park et al., 2017). MSCs-based therapies have a great promise to regenerate cartilage defects (Richardson et al. , 2010). Because of the extraordinary potential for proliferation and multipotential differentiation including chondrogenesis (Jin et al. , 2016), BMSCs have been widely studied in the mechanisms of articular cartilage repaired. However, according to the previous studies, direct application of MSCs is not efficient for their propensity to lose chondrogenic phenotype. In order to avoid inefficiency and premature degeneration, chondrogenic-related growth factors are crucial for directing and maintaining chondrogenesis in a microenvironment (Choi et al. , 2015). TGF-β1, a chondrogenesis-inducing factor, is crucial for chondrogenic differentiation of BMSCs (Shen et al., 2013; 2014). By using TGF-β1-supplemented medium, Johnstone et al. had firstly induced differentiation of human BMSCs into chondrogenic lineages (Johnstone et al. , 1998). So many scholars has been attracted to investigated the mechanism between TGF-β1 and chondrogenesis of MSCs. TGF-β1 can facilitate proliferation and maturation of chondrocytes, and improve aggregation of type II collagen and proteoglycan(Liu et al. , 2015). Tissue engineering approach, which contains isolated seed cells, degradable scaffolds and certain growth factors, seems to provide an optional solution to restore cartilage. Alginate [(C6H8O6)n], a natural polysaccharide extracted from brown algae, composed of β-D-mannuronate (M) and α-L-guluronate (G) (Growney Kalaf et al. , 2016, Lee and Mooney, 2012). It has been widely used to provide structures for transporting cells and bioactive molecules as a harmless biomaterial with lack of toxicity or immunogenicity. The Hippo pathway plays a key role in tissue regeneration(Hong et al. , 2016).And YAP, a key transcriptional co-activator, has been implicated recently in regulation of chondrogenesis, which provides new enlightenment for cartilage-repairing (Zhong et al. , 2013). Deng et al. (Deng et al. , 2016) found that Yap1 can inhibit chondrocyte hypertrophy by inhibiting Col10al expression through interaction with Runx2, a crucial regulator of chondrocyte hypertrophy. TGF-β1 can also inhibit the growth of hepatocellular carcinoma (HCC) cells by regulating the Hippo signaling pathway (Zhang et al. , 2017). And TGF-β1 in Human Embryonic Stem Cells can enhance the interactions of YAP1 and Smad to regulate cellular responses(Beyer et al. , 2013). However, it is still not clear the interactions and
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functions of the TGF-β1 and Hippo signaling pathway in the chondrogenesis of BMSCs and full-thickness articular cartilage repair. Besides, lentiviral is an optional transgenic vector due to the high infection efficiency and stable expression of target-genes (Naldini et al. , 1996). In the present study, Lentivirus-TGF-β1-EGFP was transfected in BMSCs, which were uniformly encapsulated in calcium alginate. Then BMSCs/calcium alginate constructs were implanted to cartilage defect sites in rat models. It was hypothesized that the Lentivirus-TGF-β1-EGFP transfection can enhance chondrogenesis of BMSCs to promote early-repairing of cartilage defect by TGF-β1-dependent Smad2/3 signaling and then regulate Hippo signaling to inhibit chondrocyte hypertrophy in surgically-induced full-thickness articular cartilage.
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Materials and Methods 1. Isolation and culture of BMSCs This study was approved by the Animal Care and Use Committee in Zhejiang Chinese Medical University. BMSCs were isolated from the Sprague-Dawley rats(male, 4-week-old) in a sterile environment. The bilateral femoral shafts were extracted and washed with sterile Phosphate-Buffered Saline (PBS). After removing the metaphysis to expose the bone marrow cavity, they were flushed with 10 mL serum free Dulbecco's modified eagle medium (DMEM) by 5ml syringe. Cell suspension was filtered through a 200-mesh cell sieve and centrifuged for 5min at 1000rpm. Then the cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and incubated at 37 °C and 5% CO2. By the differential time adherence method, BMSCs were separated from cells mixture. Then cells were digested using EDTA-Trypsin until reaching about 90% confluence. BMSCs were cultured and purified for three generations.
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2. Lentivirus transfection of BMSCs With the multiplicity of infection (MOI) of 100, Lentivirus-EGFP and Lentivirus-TGF-β1-EGFP (Shanghai Genechem Co., Ltd., Shanghai, China) were respectively transfected in third passage BMSCs, added with 5μl/ml polybrene. Green fluorescence was observed after 72h by using a fluorescence microscope (Olympus IX2-ILL100; Olympus Corp., Tokyo, Japan). With the cultivation in the medium above, BMSCs were divided into three groups in vitro experiment : control group(no transfection); vector group(with Lentivirus-EGFP transfection) and TGFβ1 group(with Lentivirus-TGF-β1-EGFP transfection). 3. Preparation of BMSCs/calcium alginate gel Aqueous solution of sodium alginate (2%) and calcium chloride (1%) were prepared firstly. They were sterilized by using 0.22-μm membrane filters. BMSCs, including non-transfection and Lentivirus-TGF-β1-EGFP transfection were suspended in 5ml of 2% sterile sodium alginate solution respectively at the final concentration of 1×107/ml. Then, BMSCs/sodium alginate hydrogel was dropped into calcium chloride solution to form the BMSCs/calcium alginate gel.
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4. Rat model of full thickness cartilage defect In the present study, 36 Sprague-Dawley rats (male, 12-week-old) were used. Animals were anesthetized using ketamine (80mg/kg) administered by intraperitoneal injection. With a medial parapatellar incision, the knee joints were opened to exposed the trochlear surface directly. Full thickness cartilage defects (2mm diameter 2mm deep) were drilled on the surface of trochlear groove by using an electrical drilling machine. All rats were randomly divided into three groups (n=12/group at weeks 4 and 8): model group (treated with nothing as controls), BMSCs group (treated with BMSCs/calcium alginate gel), BMSCs+TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel). After surgery, all rats were treated with postoperative antibiotic (penicillin) intramuscularly at 8U/rat for 3 days. After 4 and 8 weeks, all animals were euthanized in batches.
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5. Gross view, histology, immunohistochemistry (IHC) and histological evaluation Samples were harvested (n=6 at weeks 4 and 8) and separated freely from soft tissue and photographed for gross evaluation. International Cartilage Repair Society (ICRS) cartilage repair scores were evaluated referred to Table1 (Lee et al. , 2013).
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Table 1. ICRS cartilage repair scores Points In level with surrounding cartilage 75% repair of defect depth Degree of defect 50% repair of defect depth repair 25% repair of defect depth no repair of defect depth Complete integration with surrounding cartilage Demarcating border<1mm
Macroscopic appearance
75% repair tissue integrated, 25% with an evident border >1mm 50% repair tissue integrated, 25% with an evident border >1mm From no contact to 25% repair tissue integrated with surrounding cartilage Intact smooth surface Fibrillated surface Small, scattered fissures or cracks Several, small or few but large fissures Total degeneration of defect area
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Integration to border zone
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Standards
Scores 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0
Then they were fixed in 10% normal buffered formalin for 72h, decalcified with 14% EDTA (PH=7.4) for 30 days, dehydrated and embedded in paraffin, and cut into 3μm sections, which were stained with Alcian Blue Hematoxylin/Orange G (ABH). In order to evaluate degree of defect repair, the Mankin score system [20,21] was used.
ACCEPTED MANUSCRIPT Total 13 score was combined with structure (0-6 points), matrix (0-4 points) and cellular abnormalities (0-3 points). In the present study, lower score indicates better cartilage repair. Three independent observers performed this histopathological evaluation. IHC was performed using anti-type II collagen (Millipore, mab1330, USA), anti-pSmad2 (Abcam, ab188334, UK), anti-Runx2 (Medical & Biological Laboratories, D130-3, Japan), anti-Col10 (Abcam, ab58632, UK) and anti-YAP-1(Abcam, ab39361, UK) antibodies on the 3-μm thick tissue sections.
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6. Quantitative Gene Expression Analysis Total RNA was extracted from repaired cartilage and cells by using the RNeasy kit (Qiagen, Germany) according to the manufacturer’s instructions and measured concentration and quality by the Nanodrop 2000 (Thermo, USA). Isolated RNA was reverse transcribed to complementary DNA (cDNA) using a cDNA reverse transcription kit (Takara, Japan). QRT-PCR was performed by using an SYBR Premix EX TaqTM Ⅱ kit (Takara, Otsu, Japan). Primer sequences for GAPDH and Col2a1 are shown in Table 2.The expression level of cartilage-related genes (Col2a1) was analyzed. The ΔΔCt method was used to calculate the expression of mRNAs relative to GAPDH. All procedures were processed under RNase-free condition. Table2. Primer name and sequences for PCR analysis Primer Name Sequences GAPDH forward 5’-GAACATCATCCCTGCATCCA-3’ GAPDH reverse 5’- CCAGTGAGCTTCCCGTTCA-3’ Col2a1 forward 5’-TCCTAAGGGTGCCAATGGTGA-3’ Col2a1 reverse 5’-AGGA CCAACTTTGCCTTGAGGAC-3’
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7. Western blot Analysis Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mM phenylmethyl sulfonyl fluoride (PMSF) and protease inhibitor cocktail (Cell Signaling Technology, USA). The supernatant was collected after centrifugation at 12000 g for 30min at 4℃. Protein concentrations were detected by using a BCA Protein Assay kit (Thermo Scientific). Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, USA) incubated with different primary antibodies against β-actin (Sigma, A1978, USA), phosphorylated-Smad2/3(Cell signaling technology, #8828, USA), Smad2/3 (Cell signaling technology, #8685, USA), TGFβ1(Abcam, ab92486, UK) and YAP-1 (Abcam, ab39361, UK) overnight at 4℃. Fluorescent secondary antibodys (Licor, 926-32212 USA) were incubated with membrane in a dark room for 1h on the next day. The chemiluminescence on the membrane was detected using the he LI-COR Odyssey® scanner and software (LI-COR Biosciences, USA). 8. Statistical Analysis All data were presented as mean ± standard deviation. Statistical analyses included unpaired Student’s t-tests and one-way ANOVA test followed by the
ACCEPTED MANUSCRIPT Tukey-Kramer test. The statistical tests were performed using the software SPSS 17.0. Statistical significance was set at a 𝑃 value of <0.05.
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Results TGFβ1 is beneficial to repair cartilage defect by enhancing chondrogenesis of BMSCs. Full-thickness articular cartilage defects were made in the trochlear groove of the rat distal femur, and BMSCs/calcium alginate gel alone or transfected with TGF-β1 were implanted in the defects (Figure 1).
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Figure 1. (A) Gross views of formed BMSCs/calcium alginate gels. (B) Microscopic observation of one formed BMSCs/calcium alginate gel. Bar=2mm;.
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Macroscopically, at 4 and 8 weeks after surgery, the defect was not repaired at all in model group. Especially, friable tissue grew on the surface at weeks 8. In BMSCs group, the surface of repaired tissue was rough accompanied with a clear boundary during week 4 to 8 post-surgery. Relatively, compared to BMSCs group, the defects in BMSCs+TGFβ1 group contained more intact fillings with good integration to the surrounding normal cartilage (Figure 2A). The ICRS-cartilage repair scores in BMSCs+TGFβ1 group was significantly higher than the model group and BMSCs group (P<0.05) at 4 and 8-weeks post-op, proving better repairs than the model group and BMSCs group (Figure 2B).
ACCEPTED MANUSCRIPT Figure 2. (A)Representative gross views of repaired cartilage defects at weeks 4 and 8 in the black arrowheads. Defects were left untreated (model group) or treated with non-transfected BMSCs/calcium alginate gel (BMSCs group) and Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel (BMSCs+TGF-β1 group). (B) Evaluation of ICRS macroscopic cartilage repair scores. *P<0.05 and P<0.05 versus BMSCs+TGF-β1 group, **P<0.01 and P<0.01 versus BMSCs+TGF-β1 group.
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In order to observe the cellular structure and matrix composition between the different groups, we performed with staining of ABH and IHC for type II collagen. The histological evaluation showed parallel results. In model group, especially in 8 weeks, the defect was mainly covered with fibrous tissue. However, the defects in BMSCs+TGFβ1 group covered with more cartilage-like tissues accumulated with GAG than BMSCs group (Figure 3A). The Mankin score at weeks 4 and 8 indicated that model group were higher (8.3, 10.7) than BMSCs+TGF-β1 group (4.5, 3.5; P<0.01), and at weeks 4, BMSCs group (7.5) got higher Mankin score than BMSCs+TGF-β1 group (4.5; P<0.05). However, there is no significant difference of Mankin score between model group and BMSCs group at weeks 4(P>0.05) (Figure 3B). These data above revealed that the repaired cartilage in BMSCs+TGF-β1 group was the most similar to normal cartilage. IHC for type II collagen in each group was also performed during the weeks 4, 8 after surgery to observe chondrogenic tissue formation. The results showed that the expression of type II collagen in the repaired tissue was unobvious at both time points in model group. Repaired tissues in BMSCs group and BMSCs+TGF-β1 group stained positive for type II collagen at weeks 4 and 8 after surgery. The BMSCs+TGF-β1 group showed higher content of type II collagen than BMSCs group. By week 8, the accumulation of type II collagen in BMSCs+TGF-β1 group almost similar to the normal cartilage surrounding the defects (Figure 3C). Besides, in order to detect the cartilage-related mRNA expression of Col2a1 in the repaired tissue, quantitative real-time fluorescent PCR was used for each group. These were consistent with the histopathological results. The mRNA level of Col2a1 in BMSCs+TGF-β1 group was significantly higher than in model group and BMSCs group at the same time point (P<0.01, Figure 3D). Interestingly, Col2a1 mRNA expression in BMSCs group was higher than that in model group (P<0.01). These data above indicated that type II collagen and proteoglycan were effectively accumulated in cartilage defects treated with Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel.
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Figure 3. TGFβ1-transfected BMSCs effectively promote cartilage repairing. (A). Histological sections of repaired cartilage defects stained using Alcian Blue Hematoxylin/Orange G. Black arrowheads indicate repaired regions. Bar=3mm; (B) Mankin scores of histological repairing cartilages at defect sites; (C). Immunohistochemistry (brown) of collagen type II in repaired regions. Black arrows indicate repaired regions. Bar=1mm; (D). Total RNA was extracted from repaired tissues at weeks 4 and 8. In BMSCs+TGF-β1 group, expression of Col2a1 was significantly increased at 4 and 8 weeks. Data are presented as means ± SD. *P<0.05 and △P<0.05 versus BMSCs+TGF-β1 group, P<0.01 versus BMSCs group, **P<0.01 versus BMSCs+TGF-β1 group and P<0.01 versus model group.
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Lentivirus-TGF-β1-EGFP transfection increases activation of phosphorylation-Smad2/3 during chondrogenesis of BMSCs We analyzed TGF-β1-dependent Smad2/3 signaling activated by Lentivirus-TGFβ1-EGFP transfection to identify whether this treatment induce activation of chondrogenic differentiation through the intracellular signaling in BMSCs. IHC for pSmad2 was used to observe activation of Smad2/3 signaling during repaired tissue formation. As shown in Figure 4A, the positive cells of phospho-Smad2/3 were not apparent in Model group at the weeks 4. BMSCs alone weakly increased activation of phospho-Smad2/3. There is no significant difference between model group and BMSCs group (P>0.05). However, lentivirus-TGF-β1-EGFP transfected treatment strongly activated the phosphorylation of Smad2 at the weeks 4 (Figure 4B). It is worth noting that lentivirus-TGFβ1-EGFP transfection enhanced phospho-Smad2/3 activation in the early-period of repairing. To demonstrate the effect of the TGFβ1 on early-chondrogenic differentiation of BMSCs, we used BMSCs with non-transfected, transfected with Lentivirus-EGFP or Lentivirus-TGF-β1-EGFP and analyzed the protein expression of phospho-Smad2/3, which was specified as the TGF-β1-induced pathway to increase chondrogenic maker
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expression, such as Col2a1 (Furumatsu et al. , 2005). In vitro experiment, the Western blot results showed that the Lentivirus-TGFβ1-EGFP transfection increased phosphorylation of Smad2/3 at day 2 compared to control group and vector group (Figure 4D).
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Figure 4. Overexpression of TGF-β1 promotes the phosphorylation of Smad2/3 in BMSCs during the early repairing progress. (A). IHC of phospho-Smad2 (brown) in repaired regions. Red arrowheads: phospho-Smad2-positive cells. Bar=50μm; (B). BMSCs were observed using microscopy with normal and EGFP under the same vision at day 3 following transfection with Lentivirus-TGF-β1-EGFP. Bar=1mm; EGFP, enhanced green fluorescent protein. (C). Positive cell ratio of phospho-Smad2 was significantly increased in BMSCs+TGF-β1 group (week 4). (D). Western blot analysis demonstrated that Lentivirus-TGF-β1-EGFP was successfully transfected into BMSCs and the protein expression of pSmad2/3 was significantly increased in the BMSCs+TGF-β1 group, compared with the control and vector groups. Data are presented as means ± SD. **P<0.01 versus BMSCs+TGF-β1 group.
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TGF-β1 inhibit chondrocyte hypertrophy by regulating Hippo signaling pathway As mentioned above, TGF-β1 regulated the Hippo signaling pathway in HCC cells and YAP-1 can inhibit chondrocyte hypertrophy. In order to explore whether TGF-β1 influences the Hippo signaling pathway to inhibit hypertrophy after chondrogenic differentiation of BMSCs, we tested the expressions of YAP-1 and hypertrophic chondrocyte markers. The IHC showed that lentivirus-TGF-β1-EGFP transfected treatment obviously increased the expression of YAP-1 compared to model group and BMSCs group at the weeks 8. TGF-β1 also effectively inhibited the expressions of Runx2 and Col10 in the repaired cartilages of full-thickness articular cartilage defects rats (Figure 5A+ Figure 5B). The Western blot results in vitro also indicated that the Lentivirus-TGF-β1-EGFP transfection significantly increased YAP-1 expression at day 7 compared to control group and vector group (Figure 5C). It is proved that TGF-β1 regulated Hippo signaling pathway to inhibit chondrocyte hypertrophy by decreasing the expression of YAP-1, Runx2 and Col10.
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Figure 5. TGF-β1 decrease hypertrophic markers expression to inhibit chondrocyte hypertrophy by increasing YAP-1 expression at week8. (A). IHC of YAP-1, Runx2 and Col10 (brown) in repaired regions. (B). Positive cell ratio of YAP-1 was significantly increased in BMSCs+TGF-β1 group (week 8). (C). The protein expression of YAP-1 was significantly increased in the BMSCs+TGF-β1 group in vitro, compared with the control and vector groups; Bar=50μm; Data are presented as means ± SD. **P<0.01 versus BMSCs+TGF-β1 group.
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Discussion Clinically, cartilage damage resulted from trauma or pathologic factors is common and often reduce the patient’s quality of life (Brittberg, 2010). Most of the clinical repair methods for cartilage regeneration have favorable short-term effect, but the long-term effect is unsatisfied (Zhu et al. , 2014). Due to the poor expansion capacity of chondrocytes, autologous chondrocyte implantation is not an ideal choice for cartilage repair (Cochis et al. , 2017). In free matrix and matrix-dependent cell cultures, MSCs has shown its chondrogenic potential (Chen et al. , 2016, Johnstone et al., 1998, Noth et al. , 2002). In clinical, there are mainly two ways of using autologous MSCs to repair articular cartilage defects: intra-articular injections of MSCs (Nejadnik et al. , 2010) and surgical transplantation of MSCs-scaffold constructs (Gobbi et al. , 2014, Gobbi et al. , 2016). Tissue engineering is a promising method because it contains abundant seed cells such as MSCs which are be beneficial to cartilage repair and can avoid immunoreaction due to the source of autologous body. In this study, the BMSCs were isolated and cultured in vitro, transfected with Lentivirus-TGF-β1-EGFP and construct BMSCs/calcium alginate gel to enhance chondrogenesis of BMSCs to improve cartilage repair. We demonstrated that TGF-β1 promotes BMSCs chondrogenesis and cartilage repairing via TGF-β1-dependent Smad2/3 signaling and inhibit chondrocyte hypertrophy by regulating the Hippo signaling pathway. The results of the current study have showed the potential utility of gene-related therapy in promotion of cartilage repairing. Series of studies have proved that TGF-β1 promote MSCs to differentiate into chondrocytes, accumulate the secretion of type II collagen and proteoglycan (Liu et
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al., 2015). Our data indicate that TGF-β1 significantly enhance chondrogenesis of BMSCs in vitro and improve cartilage repairing in vivo via activation of TGF-β1-dependant Smad2/3 signaling pathway. Gross observation and histologic analysis showed that BMSCs+TGF-β1 group got the best repairing accompanied with the most smooth repairing surface, accumulation of type II collagen and proteoglycan. The results of ICRS-cartilage repair scores and Mankin score were consistent with it. The enhanced chondrogenic differentiation of BMSCs was related to increased gene expression of Col2a1. Besides, TGF-β1 can also inhibit generation of fibrous tissue in defect regions. In canonical Smad2/3 pathway, phosphorylated-Smad2 and 3 form complexes with the transcription factor Smad4. And the complexes bind specific promoters to regulate target genes (Wang et al. , 2016). Smad2/3 signaling provides equally important effects for chondrogenesis and reparative response of cartilage (Hellingman et al. , 2011). The data of Western blot and IHC proved that TGF-β1 enhances chondrogenesis of BMSCs by activating phosphorylation of Smad2/3 at the early cartilage repairing phase. Besides, Karl et al (Karl et al. , 2014) demonstrated that removed TGFβ1 from the chondrogenic medium significantly increased the hypertrophic phenotype in chondrogenic differentiating MSCs. Our data also proved that Lentivirus-TGF-β1-EGFP transfection inhibited expression of Col10a1 after chondrogenic differentiating of BMSCs during the repairing progress. Yap1, a key transcriptional co-factor in Hippo signaling pathway, promotes proliferation of early chondrocyte but interaction with Runx2, which is a crucial regulator for chondrocyte hypertrophy to suppress the expression of target genes such as Col10a1, the hypertrophy marker (Deng et al., 2016, Takeda et al. , 2001, Zheng et al. , 2003). We then analyzed whether TGF-β1 influence the expressions of hypertrophic markers of chondrogenic differentiating BMSCs via Hippo signaling pathway. We tested the expressions of YAP-1, Runx2 and Col10a1 at weeks 8 in vivo. Interestingly, the expression of YAP-1 was increased at weeks 8 when the rats were treated with lentivirus-TGF-β1-EGFP transfected BMSCs. Meanwhile, Runx2 and Col10a1 were inhibited in BMSCs+TGF-β1 group. The Western blot results in vitro also demonstrated that TGF-β1 can significantly increases YAP-1 expression after chondrogenic differentiating of BMSCs. So it means that TGF-β1 can inhibit expressions of chondrocyte hypertrophic markers via Hippo signaling. Therefore, we concluded that TGF-β1 plays two roles in cartilage repairing: 1). TGFβ1 is beneficial to chondrogenesis of BMSCs via phosphorylation of Smad2/3 to increase the expression of cartilage formation markers. 2). TGF-β1 can inhibit chondrocyte hypertrophy by decreasing hypertrophy markers expression via Hippo signaling. Conclusions We demonstrated that the implantation of Lentivirus-TGF-β1-EGFP transfected BMSCs/calcium alginate gel promoted cartilage defect repair and regeneration through chondrogenic differentiation and inhibition of hypertrophy, which are strongly associated with TGF-β1-dependent Smad2/3 signaling and Hippo signaling respectively.
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Corresponding Authors * [email protected] and [email protected] Acknowledgements This research has been partially supported by Zhejiang grants funded by Provincial Natural Science Foundation of China (Grant no. LY16H270010, LZ12H27001 and LY15H270012) Natural Science Foundation of China (Grant no.81373669, 81573994 and 81774332), the State Administration of Traditional Chinese Medicine of Zhejiang Province (Grant no. 2016ZA048, 2018ZZ011 and 2018ZA034), Major Scientific and Technological Special Project for during the Twelfth Five-year Plan Period (Grant no. 2014C13G2120082) and the Program for Zhejiang Leading Team of S&T Innovation and Key Laboratory of Zhejiang Province. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
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